Nanodosimeter based on single ion detection

ABSTRACT

A nanodosimeter device ( 15 ) for detecting positive ions induced in a sensitive gas volume by a radiation field of primary particle, comprising an ionization chamber ( 10 ) for holding the sensitive gas volume to be irradiated by the radiation field of primary particles; an ion counter system connected to the ionization chamber ( 10 ) for detecting the positive ions which pass through the aperture opening and arrive at the ion counter ( 12 ) at an arrival time; a particle tracking system for position-sensitive detection of the primary particles passing through the sensitive gas volume; and a data acquisition system capable of coordinating the readout of all data signals and of performing data analysis correlating the arrival time of the positive ions detected by the ion counter system relative to the position sensitive data of primary particles detected by the particle tracking system. The invention further includes the use of the nanodosimeter for method of calibrating radiation exposure with damage to a nucleic acid within a sample. A volume of tissue-equivalent gas is radiated with a radiation field to induce positive ions. The resulting positive ions are measured and compared with a determination of presence or extent of damage resulting from irradiating a nucleic acid sample with an equivalent dose of radiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from provisional applications Ser. No.60/200,533, titled “Nanodosimeter Based on Single Ion Detection,” filedApr. 27, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under cooperativeagreement number DAMD17-97-2-7016 with the United States Department ofthe Army. The Government has certain rights in this invention.

BACKGROUND OF INVENTION

According to modern radiobiological concepts, irreversible radiationdamage to a living cell is the consequence of multiple ionizationsoccurring within or near the DNA molecule over a distance of a fewnanometers. Such clustered ionization events can lead to multiplemolecular damages within close proximity, some of them causing strandbreakage and others various base alternations or losses, which aredifficult to repair. Unrepaired or misrepaired DNA damages typicallylead to cell mutations or cell death.

The measurement of the number and spacing of individual ionizations inDNA-size volumes can be assumed to one of the most relevant for thespecification of what can be termed “radiation quality.” By radiationquality, we refer to measurable physical parameters of ionizingradiation that best correlate to the severity of biological effectscaused in living organisms. There are a variety of practicalapplications for such measurements in radiation protection andmonitoring, as well as in radiotherapy.

The monitoring and measurement of radiation quality and theinvestigation of how it relates to the biological effects of ionizingradiation is of prime importance in many different fields includingmedicine, radiation protection, and manned space flight. For example,heavy charged particles, including protons, carbon ions, and neutronsproduce more complex radiation fields than established forms ofradiation therapy (protons and electrons). These newer forms ofradiation therapy, which are increasingly being used for the treatmentof cancer, require a careful study of radiation quality changing withpenetration depth in order to avoid unwanted side effects.

The definition of the merits and risks of these new forms ofradiotherapy requires a better understanding of the basic interactionsthese radiations have with DNA. National and international radiation andenvironmental protection agencies, e.g., the Nation Council on RadiationProtection and Measurements (NCRP) and the International Commission onRadiological Protection (ICRP), are interested in establishing newstandards of radiation quality measurements, which are based onindividual interactions of radiation with important biomolecules, mostimportantly, the DNA.

Further, radiation quality measurements are also essential to predictthe risks of human space travel. Predictions of the quality andmagnitude of space radiation exposure are still subject to largeuncertainties. Nanodosimetric measurements of space radiations orsimulated ground-based radiations may help to decrease theseuncertainties.

The measurement of local ionization clusters in DNA-size volumesrequires the development of novel nanodosimetric devices, as these wouldbe most relevant to assess DNA damage. The results of experimentalnanodosimetric studies combined with those of direct radiobiologicalinvestigations could provide a better understanding of the mechanisms ofradiation damage to cells and the reason why some DNA damage is moreserious than others leading to cancer or cell death. They would alsoprovide valuable input for biophysical models of cellular radiationdamage. There are a variety of practical applications for suchmeasurements in radiation protection and monitoring, as well as inradiotherapy.

Existing methodologies of dosimetry on a microscopic tissue-equivalentscale use microdosimetric gas detectors, for example, tissue-equivalentproportional counters (TEPCs), which measure the integral deposition ofcharges induced in tissue-equivalent spherical gas volumes of 0.2-10 μmin diameter, i.e., at the level of metaphase chromosomes and cellnuclei. They cannot be used to measure ionizations in volumes simulatingthe DNA helix. Furthermore, they provide no information about thespacing of individual ionizations at the nanometer level.

The cavity walls of these microdosimetric counters distort themeasurements, which is particularly problematic for cavity sizes belowthe track diameter. It has been suggested to use wall-lesssingle-electron counters to overcome some of these limitations. However,this method is limited by the fairly large diffusion of electrons in theworking gas and can only achieve sensitive volume sizes down to theorder of ten tissue-equivalent nanometers. The DNA double helix, on theother hand, has a diameter of 2.3 nm.

It has been suggested in the literature to overcome the limitations ofmicrodosimetric counters through the construction of a dosimeter whichwould combine the principle of a wall-less sensitive volume with theadvantage of counting positive ionization ions, which undergoconsiderably less diffusion than electrons. This would extend classicalmicrodosimetry into the nanometer domain.

This method, called nanodosimetry, is useful for radiobiology based onthe premise that short segments of DNA (approximately 50 base pairs or18 run long) and associated water molecules represent the most relevantsurrogate radiobiological targets for study. Instead of measuring thedeposition of charges directly in biological targets, nanodosimetry usesa millimeter-size volume filled with a low-density gas at approximately1 Torr pressure, ideally, of the same atomic composition as thebiological medium. Ions induced by ionizing radiation in the working gasare extracted by an electric field through a small aperture and thenaccelerate towards a single-ion counter. The sensitive volume of thedetector is defined by the gas region from which positiveradiation-induced ions can be collected using electric-field extraction.This new method would be useful for determining the biologicaleffectiveness of different radiation fields in the terrestrial andextraterrestrial environment.

The problem with prior nanodosimeters, therefore, is that they havelacked means for measurement of the energy and multi-axisposition-sensitive detection of primary particles passing through thenanodosimeter, hindering the ability to perform systematic measurementsof ionization clusters within a cylindrical tissue-equivalent volume asa function of these important parameters. Further, a method forcalibration of a nanodosimeter, e.g., correlating radiation quality withbiological damage, has been unavailable. Therefore, the goals ofnanodosimetery described above have been a long felt, but as yet unmetneed.

It would be desirable, therefore, to have a nanodosimeter which includesa particle tracking and energy measuring system that is capable ofmulti-axis position-sensitive detection of primary particles passingthrough the detector within the nanodosimeter, thereby providing theability to perform systematic measurements of ionization clusters withina cylindrical tissue-equivalent volume as a function of the position ofthe primary particle and its energy. Once configured with such aparticle tracking and energy measuring system, it would be desirable tobe able to calibrate the nanodosimeter to correlate the radiobiologicaldata of DNA damage to radiation quality, thereby relating the physics ofenergy deposition to radiobiological effects.

SUMMARY OF INVENTION

The present invention meets these needs by providing a nanodosimeterwhich includes a particle tracking and energy measuring system that iscapable of multi-axis position-sensitive detection of primary particlespassing through the detector within the nanodosimeter, and of energymeasurement of these primary particles. Using the particle tracking andenergy measuring system; a method of calibrating the nanodosimeter tocorrelate the radiobiological data of DNA damage to radiation quality,thereby relating the physics of energy deposition to radiobiologicaleffects, is also provided.

Use of a MWP detector, or preferably a silicon microstrip detector, isprovided, as well as a data acquisition system to run such ananodosimeter, and thereby process primary particles and secondaryionizations on an event-by-event basis. The provided system is able tomeasure the energy of primary particle, and detect the location ofprimary particles, and allow on-line reduction of very large statisticalsamples, capable of simultaneous detection and counting of particles

The apparatus and method measure individual ions produced by ionizingparticles in a wall-less, low-pressure gas volume, which simulates abiological sample of nanometer dimensions. Changing pressure conditions,the size of the sensitive volume can be modified. Modifying theelectrical field configuration inside the detector, also the shape ofthe sensitive volume can be adjusted. The detector registers the numberof ions produced in the sensitive volume as well as their spacing alongthe principal axis of the sensitive volume; both quantities are believedto be important for the biological effectiveness of terrestrial andextraterrestrial radiation. The new detector can be used to provideinput data of biophysical models that can predict the biologicalefficiency or quality of the radiation under investigation. Anothernovel aspect of the device is that almost any gas composition can beused in order to study radiation effects in the various subcompartmentsof the biological system, e.g., water and DNA.

BRIEF DESCRIPTION OF DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings, where:

FIG. 1A is a cross-sectional diagram of a front view of a nanodosimetercapable of being used with the present invention;

FIG. 1B is a cross-sectional diagram of a side view of the nanodosimeterof FIG. 1A;

FIG. 2 is a conceptual diagram of the single ion counting method ofnanodosimetry used in the present invention;

FIG. 3 is a cross-section diagram of the ionization cell and high vacuumchamber of the nanodosimeter of FIG. 1A;

FIG. 4 is graph of calculated sensitive volume configurations as used inthe present invention;

FIG. 5 is a graph of an example of a recorded ionization event using thenanodosimeter of FIG. 1A;

FIG. 6 is a schematic diagram of a nanodosimeter incorporating amulti-axis particle tracking system according to one embodiment of thepresent invention;

FIG. 7 is a schematic diagram of a nanodosimeter incorporating aparticle tracking and data acquisition system according to anotherembodiment of the present invention;

FIG. 8 is a schematic diagram of a nanodosimeter incorporating aparticle tracking and data acquisition system according to furtherembodiment of the present invention;

FIG. 9 is a pictorial flow chart representing a calibration methodaccording to one embodiment of the present invention;

FIG. 10 is a pictorial flow chart representing a calibration methodaccording to another embodiment of the present invention;

FIG. 11 is a graph of an example of a frequency distribution of recordedevents by strip number, according to one embodiment of the presentinvention;

FIG. 12 is a flow chart show the method of calibration of thenanodosimeter of FIG. 1A, comprising one of the embodiments of theparticle tracking system, to biological damage according to the presentinvention;

FIG. 13 is a graph showing calibration of the Time-Over-Threshold ASICin one embodiment of the particle tracking system, in which TOT is afunction of the input charge in multiples of the charge deposited by aminimum ionizing particle (MIP); and

FIG. 14 is a graph showing Predicted Time-over-Threshold (TOT) signal inone embodiment of the particle tracking system, as a function of theproton energy.

DETAILED DESCRIPTION

The present invention will be better understood with respect to FIGS.1-11 that accompany this application.

FIGS. 1A and 1B show detailed drawings of a nanodosimeter 15 that iscapable of being used with the present invention, comprising anionization chamber 10 holding a low-pressure gas, into which radiationis injected either from a built-in a particle source 16 which is incommunication with the ionization chamber, or from any externalradiation source after passing through an entrance window 17, a detector11 and an ion counter 12 for counting ionized particles. A differentialpumping system comprising two pumps 14 and 15 is also provided tomaintain a relatively low pressure (e.g., a high vacuum) in the chamberholding the ion counter, while maintaining a higher pressure within theionization chamber. Any suitable radiation source emitting ionizedcharged particles with sufficient energy to penetrate window 17 can beused, as will be evident to those skilled in the art. Any suitabledetector 11 can be used, such as a scintillator-photomultiplier tube(PMT) combination, as will also be evident to those skilled in the art.

FIG. 2 is a conceptual diagram of the single ion counting method used innanodosimeter 15, showing how a wall-less sensitive volume 21 can beformed in ionization chamber 10 filled with a low-pressure density gas23 of approximately 1 Torr. An energetic ionizing particle 22 traversingionization chamber 10 induces ionizations around its track, directly andthrough the mediation of δ electrons 24. Radiation-induced positive ionsdrift under a relatively weak electric field Esub1 25 of about 60-100V/cm through a narrow aperture (about 1 mm diameter) at the bottom ofthe ionization chamber 10 toward the ion counter 12. Below the aperturethe ions experience a much stronger electric field Esub2 26 of about1500-2000 V/cm. The electric field strength and the diameter of theaperture define the lateral dimensions of a wall-less sensitive volumeabove the aperture from which the ions can be extracted with highefficiency. By changing the pressure inside the ionization chamber onecan make further adjustments to the size of the sensitive volume, aswill be evident to those skilled in the art. Since positive ions diffusemuch less than electrons, sensitive volumes of about 0.1-4.0 nm tissueequivalent diameter and 2-40 nm tissue-equivalent length can be achievedwith this method. By applying a time window during which ions arecounted one can further define a subsection of the sensitive volume fromwhich ions are counted.

Various cellular subsystems, most importantly water and DNA, can besimulated by using gases of different composition. As standard gas, onemay use propane. As the low-energy ions do not undergo gasmultiplication there are no limits on the gas 23 to be investigated.

FIG. 3 shows the actual design of the ionization chamber 10 and iondrift optics 35 of nanodosimeter 15. The upper electrode 31, which is ata positive potential of 300-500 Volts produces the drift field withinthe ionization chamber. A gold-plated aperture plate 32, which is atground potential, contains an opening aperture 33 of about 1 mmdiameter. Different aperture sizes may be used to adjust the width ofthe sensitive volume. Electrodes in ion drift optics 35 and a metal cone34 generate the electric field below the aperture, which focus andaccelerate the ions toward the cathode of the ion counter 12. Theelectrodes of the ion drift optics and metal cone are held at a negativepotential of about 450 Volts, while the ion counter cathode is at anegative potential of about 7,000 to about 8,000 Volts.

FIG. 4 shows calculated ion collection efficiency maps 41 and 42 of thesensitive volume for a field condition of temperature of 280 degrees K.,a homogeneous electric field of 100 V/cm, and focusing field near theaperture 33, where map 41 is map 42 expanded for clarity. The maps arebased on Monte Carlo studies of individual ion trajectories and measuredion diffusion parameters. The maps take into account actual electricfield inhomogeneities. By changing the electric field strengths one canchange the length and lateral diameter of the sensitive volume. In afurther embodiment, the placement of additional electrodes in thevicinity of the sensitive volume and application of appropriate positivepotentials enables the shape of the sensitive volume to be influenced.For example, the “candle-flame” shaped volume shown in FIG. 4 can bechanged to a cylindrical volume by applying higher field strengths inthe upper part of the volume.

Individual ions collected from the sensitive volume are counted with avacuum-operated electron multiplier 12 of a type usually employed inmass spectroscopy. The model 14180HIG active film multiplier, SGE, or anequivalent, would be suitable. The counter generates fast signals frommultiplied secondary electrons originating from the interaction of theaccelerated ions with the multiplier cathode.

The ion counter 12 requires a vacuum in the order of 10⁻⁵ Torr.Maintaining this vacuum against the pressure of about 1 Torr in theionization chamber 10 requires use of a differential pumping systemconsisting of two powerful turbo-molecular pumps. Pumps suitable for thepurpose include the Varian Vacuum Technologies, Inc., models V250 forpump 14, and model V550 for pump 13, as shown in FIGS. 1A and 1B.

FIG. 5 shows an example of an ion trail spectrum produced by thenanodosimeter 15 of an alpha particle traversing the sensitive volume21, where the x axis is in microseconds and the y axis is in millivoltsmeasured by the ion counter 12.

A primary particle detection system which provides identification ofsingle particle events must be added to the nanodosimeter 15.Furthermore, this provides for the measurement of the arrival time ofthe ions relative to the primary particle passage, thereby enabling thespatial localization of the ionization event along the principlesymmetry axis of the sensitive volume. Due to the low mobility of theions, the events are well separated in time. It has been shown that aspatial resolution of 1 nm tissue equivalent length can be achieved.

FIG. 7 shows one embodiment of such a particle detection system,schematically diagraming how a detector is embedded into the triggeringand data acquisition system. The implementation of a particle detectionsystem tracking system enables the measurement of ionization clusters inthe sensitive volume 21 for each primary particle event 73. The primaryparticle event 73 is reconstructed from the signals of particlesensitive detectors located in front and behind the sensitive volume. Inthis embodiment, three fast plastic scintillators (BC 408, Bicron), twoof which are located at the front 761 and 751 and one at the rear end771 of the ionization chamber 10, register primary particles that enterthe ionization chamber 10 and pass through it. The down-stream front-endscintillator 751 contains an opening 750 of a specified shape and isused in anticoincidence to the up-stream front-end scintillator 751 toselect the cross-sectional area of particle detection. Photomultipliertubes (PMTs) 762, 752 and 772 register the light signals provided by thescintillators. The PMT signals 78 are then processed by fast front-endelectronics (preamplifiers and discriminators) 79 and sent to dataacquisition boards (PCI 6602, PCI6023E, National Instruments) via aninterface board, which provides fast NIM signal conversion to TTL/CMOSsignals. The data acquisition boards 793 perform time-to-digitconversion of the arrival times of each signal and amplitude-to-digitconversion of the rear scintillator signal, which contains informationabout the energy of the primary ionizing particle. The digital data aresent along a PCI bus 791 to a dedicated data acquisition PC 792, wherethey are processed, displayed and stored.

In this embodiment, the data acquisition process can also besynchronized to gate signals provided by the external radiation source,for example, a synchrotron which delivers particles in form of spillswith a complex time structure.

FIG. 8 shows a further embodiment of a position-sensitive triggeringsystem. For clarity, the ion counter 12 is not shown in this figure.This embodiment uses, within the ionization chamber 10, a multiwireproportional chamber (MWPC) 80 as used in high-energy physicsexperiments, upstream of the sensitive volume and a double-sided siliconmicrostrip detector (e.g., S6935, Hamamatsu Corp.) 81 downstream of thesensitive volume. In this configuration, the distance between thesensitive volume 21 and the MWPC 80 is in the order of 8 cm, whereas itis only about 3 cm between the sensitive volume 21 and the microstripdetector 81, With this configuration, a spatial resolution of the trackposition in the plane of the sensitive volume is in the order of 100 μm(0.1 mm). It has been known from high energy physics experiments thatsilicon microstrip detectors can be used for precise tracking of chargedparticles, but have not been implemented in nanodosimetry.

Very large statistical samples must be accumulated with thenanodosimeter to detect rare high-order ionization events. Readoutschemes for on-line reduction of such samples, utilizing a digitalsignal processor located on front-end boards 84 and 85 and embeddedcomputer networks 83, as shown in FIG. 8, are now widely used inhigh-energy physics experiments, but have not been implemented innanodosimetry. Using the system shown in FIG. 8, signals 86 comprisinginformation on energy, and multi-dimensional position of the primaryparticle can be passed to the DAQ computer 792 for processing.

FIG. 6 shows a further embodiment of a position-sensitive triggering andenergy measurement system integrated into the nanodosimeter 15. Thisembodiment comprises two silicon strip detector modules 61 and 62 thatconvey the X- and Y-position of the particle 22 relative to thesensitive volume with a resolution that is determined by the strippitch, and which is usually better than 0.2 mm. In addition, the siliconstrip detectors can measure the energy deposited by each primaryparticle across the depletion layer of the silicon crystals, thusproviding information about the energy and LET of the primary particleover a wide range of particle energies. Using the system shown in FIG.6, signals comprising information on energy, and multi-dimensionalposition of the primary particle can be passed to the DAQ computerthrough interface board 63 for processing.

FIG. 11 shows, as an example of the position-sensitive systemperformance of the silicon strip embodiment, a hit-strip distribution110 providing particle position information, and the time-over-thresholddistribution 111 representing the energy deposition distribution of a 40MeV proton beam collimated to 1.5-mm width. The hit-strip distributionclearly demonstrates the high spatial resolution of particle coordinatemeasurements.

In this embodiment, the front silicon-strip detector module comprisestwo single-sided silicon micro-strip detectors with orthogonal striporientation, and the back detecor module comprises one double-sidedsilicon micro-strip detector located behind the sensitive volume. Thisarrangement of detectors provides information about the primary particletrack from the strip-hit information as well as the particle's energyover a wide range of energies. This allows quantifying the nanodosimeterinformation as a function of the primary particle energy and position.

For the readout of the fast silicon detector signals, it is preferableto use a low-noise, low-power front end ASIC, such as was developed forthe GLAST mission, in which the input charge is measured through thepulse width, i.e., as a time-over-threshold (TOT) signal, over a largedynamic range. An example of the measured electronic calibration of thechip TOT vs. input charge (in units of charge deposited by a minimumionizing particles, MIP) is shown in FIG. 13.

FIG. 14 shows an expected TOT signal vs. the energy of the primaryprotons incident on a silicon micro-strip detector using the threedetector (two single-sided silicon micro-strip/one double-sided siliconmicro-strip) embodiment. The method of determining the energy of theproton from its specific energy deposition using the TOT signal of asingle detector is expected to be viable for proton energies above about10 MeV and below about 3 MeV. The proton energy can be measured uniquelyat energies above 10 MeV and below 3 MeV. However, in the energy rangeof about 3-15 MeV, protons deposit a significant fraction of theirenergy in the silicon detector, so that the TOT signal of at least oneof the three detectors will be within the measurable range, and thuswill provide sufficient information to reconstruct the energy of theparticle passing the ionization chamber. At higher energies, i.e., aboveabout 15 MeV, the TOT signal is a relatively shallow function ofincident proton energy, but for these energies all three detectors willprovide a measurement, thereby reducing the measurement uncertainty.

Monte Carlo calculations with low energy proton beams can be used totest a relationship such as shown in FIG. 14, and to determine theresolution of this energy determination method. Since multiplescattering of protons in the low-pressure gas volume of the detector isminimal, the position resolution is mainly determined by the pitch ofthe micro-strip detectors.

Each of the described embodiments for a position-sensitive trackingsystem requires a data acquisition system (DAQ), that receives inputfrom the ion counter 12, primary particle trigger signals either fromthe built-in particle tracking system or from scintillators, anaccelerator start signal when used with a synchrotron accelerator, andposition and energy-deposition data from the particle-tracking system.The DAQ system preferably uses fast PCI technology which receives andsends data from and to an interface board 63 with reference to FIG. 6.The DAQ system coordinates the readout of all data signals and performsonline and offline data analysis.

In another embodiment, the present invention is a method of correlatingthe response of the nanodosimeter with the presence or extent of damageto a nucleic acid within a sample. In a preferred embodiment, thenucleic acid containing sample is an in-vitro solution of plasmid DNA.In other embodiments, the DNA is viral, chromosomal, or from aminichromosome.

With reference to FIG. 12, the method typically includes specifying atissue-equivalent sensitive volume of a tissue-equivalent gas 120. Thetissue equivalent sensitive volume is typically selected to model aparticular tissue equivalent volume, such as a discrete length of adouble stranded DNA. In the current embodiment, the typical sensitivevolume can be specified to be between about 0.1 nm and about 4 nmtissue-equivalent in diameter and between about 2 nm and up to about 40nm in tissue-equivalent length. In one embodiment, the sensitive volumeis the tissue-equivalent sensitive volume is between about 0.2 nm³ andabout 500 nm³. Preferably, the tissue-equivalent sensitive volume isbetween about 20 nm³ and about 100 mm³. The optimal sensitive volumesize and gas composition is that which gives the highest degree ofcorrelation between measured DNA lesions and those predicted fromnanodosimetric data.

The method further comprises irradiating the tissue-equivalent gas andthe sample with a radiation field 121. Preferably, the nucleic acidcontaining sample is exposed to a substantially equivalent quality ofradiation that is measured by the nanodosimeter 15. The plasmid istypically dissolved in an aqueous solution that simulates the cellularenvironment such as, for example, a solution including glycerol and abuffer. This is done to reproduce the diffusion distance of OH radicalswith a living cell. Preferably, the sample is irradiated with a range ofdoses in order to establish a dose-response relationship. Preferably,the DNA concentration and range of irradiation doses are selected suchthat each plasmid will, on average, contain about one DNA damage ofvariable complexity. For example, irradiation of a plasmid sample havinga concentration of 1 mg/ml with a dose of about 10 Gy of low-LETradiation will result, on average, in one single stranded break for eachplasmid.

In a preferred embodiment, the number of positive ions induced withinthe tissue-equivalent sensitive volume by the radiation field isdetected 122 using an embodiment of the nanodosimeter with particletracking and energy measuring system described herein.

The frequency distribution of damages of variable complexity to thenucleic acid within the sample is compared with the frequencydistribution of variable clusters of positive ions induced within thetissue-equivalent sensitive volume. By damage of variable complexity werefer to base damages (B) or strand breaks (S) occurring on eitherstrand of the DNA and ranging from a single damage site to multiplecombinations of these damages.

One embodiment of the calibration assay is illustrated in FIG. 10. Inthis embodiment, each sample includes a thin film of an aqueous solutionof plasmid DNA 91. The sample is exposed to the same radiation qualityas the nanodosimeter. The irradiated plasmid sample is optionallytreated with a base-excision enzyme 124 such as endonucleaseformamidopyrimidine-DNA N-glycosylase (FPG) or endonuclease III. Theseenzymes transform base damages in the DNA of irradiated plasmids intostrand breaks. Damages that contain at least one strand break located incomplementary strands in close proximity to each other are convertedfrom a closed supercoiled form into a linear form. Damages that containat least one strand break on only one strand will be transformed into arelaxed open circle.

The different physical states of plasmids (supercoiled, open circle,linear) are separated by agarose gel electrophoresis and quantifiedafter staining with a fluorescent dye 123. The calibration assay allowsone to distinguish and to measure 125 the absolute or relative frequencyof the following types of DNA lesions: lesions that contain at least onestrand break on one strand but not on the other strand (S0); lesionsthat contain at least one base damage on one strand but not on the otherstrand (B0); lesions that contain at least one strand break oncomplementary strands (SS); lesions that contain at least one basedamage on complementary strands (BB); and lesions that contain at leastone base damage on one strand and at least one strand break on the otherstrand (SB).

According to another embodiment of the calibration assay shown in FIG.9, thin films of supercoiled plasmids conferring antibotic resistanceand a reporter gene such as β-galactosidase are exposed to the sameradiation quality as the detector. Plasmids that contain DNA damages ofvariable complexity are separated from each other and undamaged plasmidsby gel electrophoresis. After separation, the damaged plasmids areextracted from the gel and incubated with a mammalian repair extract 126or a Xenopus laevis oocyte extract for several hours to allow DNA damageto be repaired. After incubation, the plasmids are transfected intoantibiotic-sensitive bacterial host cells 127 and the bacteria are grownin the presence of the antibiotic to select for successfully transformedbacteria. The reporter gene of the plasmid is used to detect misrepairedDNA damage. Cells containing the intact gene produce a colored dye whenincubated with the indicator. This compound is colorless, unless cleavedby β-galactosidase. Colonies that contain a non-functional reporter geneare not colored.

In this way, one can measure the fraction of unrepaired or misrepaireddamage in a given amount of DNA for different radiation qualities, andby comparison with nanodosimetric event spectra 128, identify ionizationevents leading to mis-reparable DNA damage.

In another aspect of the invention, the probability that a singleionization event proximal to a nucleic acid will result in a singlestrand break or a base damage is determined by the calibration assay.From this, the frequency of the each type of nucleic acid lesion iscalculated for ionization clusters of a given size. The calculatedfrequency of particular nucleic acid lesions for ionization clusters ofa discrete size is compared with the frequency distribution ofionization clusters measured with the nanodosimeter to predict theabsolute and relative frequency of each type of nucleic acid lesion.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. For example, in alternative embodiments the invention includesa method for determining a dose of radiation for radiation therapy usingthe procedure, a method of predicting death or mutation in a livingcell, a method of modeling the effect of radiation in a living cell, amethod evaluating radiation risk for manned space missions, andassessment of radiation exposure of aircraft crew and frequent flyers.The present invention has many potential applications to various areasincluding but not limited to planning and optimizing of radiationtherapy with charged particles, design and evaluation of radiationshielding, radiation protection, monitoring of occupational and otherterrestrial radiation environments. Therefore, the scope of the appendedclaims should not be limited to the description of the preferredversions described herein.

One particularly important and novel application of the nanodosimeter isthe determination of W, the average energy required to produce an ionpair in gases as a function of particle energy. More accurately, W isthe quotient of E and N, where N is the mean number of ion pairs formedwhen the initial kinetic energy, E, of a charged particle is completelydissipated in the gas. While W is known with good accuracy only for alimited number of particle types and energies, accurate knowledge of theenergy dependence of W is highly desirable both for basic understandingof dosimetric theory and for application in medical dosimetry. Forexample, accurate determination of the dose delivered in neutron orproton therapy requires mapping of the energy dependence of W forprotons and heavy recoil ions over a wide range of energies with anaccuracy of better than 2%. This goal has currently not beenaccomplished.

The nanodosimeter can be used to measure the differential value, w(E),of the mean energy necessary to produce an ion pair relative to a knownvalue W(E_(ref)) at a reference energy E_(ref). The differential value wis defined as the quotient of dE by dN, where dE is the mean energy lostby a charged particle of energy E traversing a thin gas layer ofthickness dx, and dN is the mean number of ion pairs formed when dE isdissipated in the gas. Alternatively, one may express w as a function ofthe stopping power S(E)=dE/dx of the gas, which is usually known withgood accuracy:w(E)=S(E)/dN·dx

With the particle tracking system of the nanodosimeter one can selectprimary particles with the reference energy E_(ref) and a given energy Ethat pass the sensitive volume of the nanodosimeter at a given distancey from the aperture. The ratio of N₁(E_(ref)) and N₁(E), the averagenumber of nanodosimetric ion counts for primary particle energiesE_(ref) and E, can then be used as a good approximation for the ratio ofdN(E_(ref)) and dN(E), thusw(E)/w(E _(ref))=S(E)/S(E _(ref))N₁(E _(ref))/N₁(E)

All features disclosed in the specification, including the claims,abstracts, and drawings, and all the steps in any method or processdisclosed, may be combined in any combination, except combinations whereat least some of such features and/or steps are mutually exclusive. Eachfeature disclosed in the specification, including the claims, abstract,and drawings, can be replaced by alternative features serving the same,equivalent or similar purpose, unless expressly stated otherwise. Thus,unless expressly stated otherwise, each feature disclosed is one exampleonly of a generic series of equivalent or similar features.

Any element in a claim that does not explicitly state “means” forperforming a specified function or “step” for performing a specifiedfunction, should not be interpreted as a “means” or “step” clause asspecified in 35 U.S.C. § 112.

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure.

1-49. (canceled)
 50. A nanodosimeter device for detecting positive ionsinduced in a sensitive gas volume by a radiation field of primaryparticles, comprising: an ionization chamber for holding the sensitivegas volume to be irradiated by the radiation field of primary particles,the ionization chamber having an aperture opening; an ion counter systemconnected to the ionization chamber for detecting the positive ions, theion counter system having an ion counter in communication with theaperture opening; a particle tracking system having a position-sensitivedetector for detecting the primary particles passing through thesensitive gas volume; and a data acquisition system that receives andcorrelates data from the ion counter system and the particle trackingsystem.
 51. The nanodosimeter of claim 50, wherein the particle trackingsystem is capable of multi-axis position-sensitive detection of theprimary particles passing through the sensitive gas volume.
 52. Thenanodosimeter of claim 50, wherein the particle tracking system furthercomprises an energy measurement system for measuring the energy of theprimary particles passing through the sensitive gas volume.
 53. Thenanodosimeter of claim 50, further comprising a radiation source incommunication with the ionization cell chamber, for injection of theradiation field of primary particles into the ionization cell chamber.54. The nanodosimeter of claim 53, wherein the radiation source is asource of α particles.
 55. The nanodosimeter of claim 53, wherein theradiation source is a source of ionizing particles.
 56. Thenanodosimeter of claim 53 wherein the radiation source is anaccelerator.
 57. The nanodosimeter of claim 56, wherein the acceleratorcomprises a synchrotron.
 58. The nanodosimeter of claim 50, wherein theposition-sensitive detector comprises a plurality of scintillators andphotomultiplier tubes.
 59. The nanodosimeter of claim 50, wherein theposition-sensitive detector comprises a silicon microstrip and amultiwire proportional chamber.
 60. The nanodosimeter of claim 50,wherein the position-sensitive detector comprises a plurality of siliconmicrostrips.
 61. The nanodosimeter of claim 50, wherein the sensitivegas volume comprises propane.
 62. A method for measuring positive ionsinduced by a radiation field of primary particles, comprising the stepsof: providing a tissue-equivalent gas; determining a tissue-equivalentsensitive gas volume of the tissue-equivalent gas; irradiating thesensitive gas volume with the radiation field; detecting the positiveions induced by the radiation field; tracking the primary particles thatpass through the sensitive gas volume; and correlating data regardingthe positive ions and primary particles.
 63. The method of claim 62,further comprising the step of scaling the data from the nanodosimeterfor the sensitive gas volume to a DNA-size volume.
 64. The method ofclaim 62, further comprising the steps of: selecting primary particlesusing the particle tracking system with a reference energy E_(ref) and agiven energy E that pass the sensitive gas volume; calculating a ratioof N₁(E_(ref)) and N₁(E), which are the average number of nanodosimeterion counts for primary particle energies E_(ref) and E, respectively;using the ratio of N₁(E_(ref)) and N₁(E) as an approximation for theratio of dN(E_(ref)) and dN(E); and computing the differential valuew(E) according to the formulaw(E)/w(E _(ref))=S(E)/S(E _(ref))N ₁(E _(ref))/N ₁(E).
 65. A method forcalibrating radiation exposure from a first radiation field with thepresence or extent of damage to a nucleic acid within a sample, themethod comprising the steps of: providing a tissue-equivalent gas;determining a tissue-equivalent sensitive gas volume of thetissue-equivalent gas; irradiating the tissue-equivalent gas and thesample with the first radiation field; calculating the number ofpositive ions induced within the sensitive gas volume by the firstradiation field; detecting the presence or extent of damage to thenucleic acid within the sample following irradiation with a secondradiation field; and comparing and correlating the extent of damage tothe nucleic acid within the sample with the calculated number ofpositive ions induced by the first radiation field.
 66. The method ofclaim 65, wherein the second radiation field is equal to the firstradiation field.